PROJECT SUMMARY/ABSTRACT Gamma/delta (??) T cells are innate-like lymphocytes that express unconventional antigen receptors (?? TCR) on their surface. Unlike conventional ?? T cells, they do not recognize peptide-MHC antigens but are instead activated by invariant stress-inducible receptors and host/pathogen-associated metabolites. Most ?? T cells in human peripheral blood express the V?9V?2 TCR and are activated by phosphoisoprenoid (PiP) metabolites produced by medically important microbes, viruses and cancer cells. V?9V?2 T cell activation by PiPs is TCR- dependent, and requires contact with PiP-producing target cells that express the B7-family protein butyrophilin 3A1 (BTN3A1). However, the molecular mechanisms by which PiPs and BTN3A1 initiate signaling and activate V?9V?2 T cells remain unresolved. Our overall objective in this proposal is to identify the cell biological and molecular basis of PiP-dependent V?9V?2 T cell activation. In ?? T cells, antigen-induced signaling occurs at the cell-cell contact interface between T cells and antigen-bearing target cells, known as the immunological synapse. We propose to employ cutting edge approaches that include super-resolution fluorescence imaging, electron microscopy/tomography (EM), CRISPR/Cas9-based genomic editing, and proximity-based chemical tagging, to test our central hypothesis: that PiP-mediated V?9V?2 T cell activation occurs through a uniquely configured immunological synapse, in which TCR, accessory receptors (including BTN3A1), cytoskeletal elements, and lipid domains act in cis and across the synapse to initiate signaling. Our substantial preliminary investigations establish the feasibility of our approach and point to a need for `close contacts' and synaptic TCR/phosphatase segregation for effective PiP-dependent T cell activation. Surprisingly, we find that expression of BTN3A1 is required in both target cells and T cells, pointing to the possibility of an unsuspected trans-synaptic interaction of BTN3A1. To test our central hypothesis, we propose two Specific Aims: 1.) Using super-resolution imaging and EM, in conjunction with biochemical, signaling and functional assays, establish the PiP-dependent molecular organization, membrane topography, and key signaling events at the V?9V?2 T cell synapse; and 2.) Using genome-editing and RNAi approaches, combined with BTN3A1 structure/function mutagenesis, establish the role of PiP-induced changes in cell-surface organization of BTN3A1 in V?9V?2 T cell activation. Our approach is innovative as it uses cutting-edge imaging and biochemical methods to test a novel model for PiP-mediated V?9V?2T cell activation. The proposed research is significant as it will identify the fundamental biochemical, biophysical and cell biological requirements for V?9V?2 T cell activation, and provide a mechanistically-based framework to harness ?? T cell immunity for cellular immunotherapy.